Genji Qin

NPY genes and AGC kinases define two key steps in auxin-mediated organogenesis in Arabidopsis

Auxin is an essential regulator of plant organogenesis. Most key genes in auxin biosynthesis, transport, and signaling belong to gene families, making it difficult to conduct genetic analysis of auxin action in plant development. Herein we report the functional analysis of several members of 2 gene families (NPY/ENP/MAB4 genes and AGC kinases) in auxin-mediated organogenesis and their relationships with the YUC family of flavin monooxygenases that are essential for auxin biosynthesis. We show that 5 NPY genes (NPY1 to NPY5) and 4 AGC kinases (PID, PID2, WAG1, and WAG2) have distinct, yet overlapping, expression patterns. Disruption of NPY1 does not cause obvious defects in organogenesis, but npy1 npy3 npy5 triple mutants failed to make flower primordia, a phenotype that is also observed when AGC kinase PID is compromised. Inactivation of YUC1 and YUC4 in npy1 background also phenocopies npy1 npy3 npy5 and pid. Simultaneous disruption of PID and its 3 closest homologs (PID2, WAG1, and WAG2) completely abolishes the formation of cotyledons, which phenocopies npy1 pid double mutants and yuc1 yuc4 pid triple mutants. Our results demonstrate that NPY genes and AGC kinases define 2 key steps in a pathway that controls YUC-mediated organogenesis in Arabidopsis.

d- myo -Inositol-3-Phosphate Affects Phosphatidylinositol-Mediated Endomembrane Function in Arabidopsis and Is Essential for Auxin-Regulated Embryogenesis

This work reveals the significant roles of myo-inositol in Arabidopsis embryogenesis. Experiments show that depletion of myo-inositol by knocking out multiple MIPS genes, which encode d-myo-inositol-3-phosphate synthase, could affect the membrane trafficking via the phosphatidylinositol metabolism by leading to altered auxin distribution in developing embryos. In animal cells, myo-inositol is an important regulatory molecule in several physiological and biochemical processes, including signal transduction and membrane biogenesis. However, the fundamental biological functions of myo-inositol are still far from clear in plants. Here, we report the genetic characterization of three Arabidopsis thaliana genes encoding d-myo-inositol-3-phosphate synthase (MIPS), which catalyzes the rate-limiting step in de novo synthesis of myo-inositol. Each of the three MIPS genes rescued the yeast ino1 mutant, which is defective in yeast MIPS gene INO1, and they had different dynamic expression patterns during Arabidopsis embryo development. Although single mips mutants showed no obvious phenotypes, the mips1 mips2 double mutant and the mips1 mips2 mips3 triple mutant were embryo lethal, whereas the mips1 mips3 and mips1 mips2+/− double mutants had abnormal embryos. The mips phenotypes resembled those of auxin mutants. Indeed, the double and triple mips mutants displayed abnormal expression patterns of DR5:green fluorescent protein, an auxin-responsive fusion protein, and they had altered PIN1 subcellular localization. Also, membrane trafficking was affected in mips1 mips3. Interestingly, overexpression of PHOSPHATIDYLINOSITOL SYNTHASE2, which converts myo-inositol to membrane phosphatidylinositol (PtdIns), largely rescued the cotyledon and endomembrane defects in mips1 mips3. We conclude that myo-inositol serves as the main substrate for synthesizing PtdIns and phosphatidylinositides, which are essential for endomembrane structure and trafficking and thus for auxin-regulated embryogenesis.

Targeted mutagenesis in rice using CRISPR-Cas system.

Genome editing of model organisms is essential for gene function analysis and is thus critical for human health and agricultural production. The current technologies used for genome editing include ZFN (zinc-finger nuclease), meganucleases, TALEN (Transcription activa-tor-like effector nucleases), etc. [1]. These technologies can generate double stranded breaks (DSBs) to either disrupt gene function through generation of premature stop codons by non-homologous end joining (NHEJ) pathway, or to facilitate gene targeting through homolo-gous recombination (HR) with an incoming template. Recently, a new technology for genome editing, CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated) systems, has been developed [2]. CRISPR/Cas systems are adaptive defense systems in prokaryotic organisms to fight against alien nucleic acids [3]. The spacer sequences acquired from foreign DNA are positioned between host repeats, and transcribed together as CRISPR RNA (crRNA). In the type II CRISPR system, a single nuclease Cas9, guided by a dual-crRNA:tracrRNA, is sufficient to cleave cog-nate DNA homologous to the spacer [2]. Efficient cleav-age also requires the presence of protospacer adjacent motif (PAM) 5′-NGG-3′ following the spacer sequence. The dual-crRNA:tracrRNA has been further streamlined to a single RNA chimera, called sgRNA (single guide RNA) [2]. Compared with protein-guided technologies, CRISPR/Cas system is much easier to implement, as only short guide RNAs need to be customized to target the genes of interest. Up to now, the CRISPR/Cas system has been successfully applied to efficient genome editing in many eukaryotic organisms including human [1], mice [4], zebra fish [5], fly [6], worm [7], and yeast [8]. However, the application of CRISPR/Cas system in plants has not been reported. Rice (Oryza sativa L.) is a major staple crop in the grass family (Poaceae), feeding half of the worlds population. Rice is also used as a model monocot plant for biological studies because it has a relatively small genome compared to other cereal crops and is easy to be manipulated genetically. We demonstrate in this study that the CRISPR/Cas technology can achieve efficient targeted mutagenesis in transgenic rice. Our work paves the way for large-scale genome editing in rice, which is important for quality improvement and yield increase of rice. To accommodate the CRISPR/Cas system to Agro-bacterium-mediated plant transformation, we designed Gateway TM binary T-DNA vectors for co-expression of CAS9 and guide RNA (either sgRNA or dual-crRNA:tracrRNA, see Figure 1A). Gene-specific spacer sequence was cloned into entry vectors for expression of guide RNA (Supplementary information, Figure S1 and …

An Indole-3-Acetic Acid Carboxyl Methyltransferase Regulates Arabidopsis Leaf Development

Auxin is central to many aspects of plant development; accordingly, plants have evolved several mechanisms to regulate auxin levels, including de novo auxin biosynthesis, degradation, and conjugation to sugars and amino acids. Here, we report the characterization of an Arabidopsis thaliana mutant, IAA carboxyl methyltransferase1-dominant (iamt1-D), which displayed dramatic hyponastic leaf phenotypes caused by increased expression levels of the IAMT1 gene. IAMT1 encodes an indole-3-acetic acid (IAA) carboxyl methyltransferase that converts IAA to methyl-IAA ester (MeIAA) in vitro, suggesting that methylation of IAA plays an important role in regulating plant development and auxin homeostasis. Whereas both exogenous IAA and MeIAA inhibited primary root and hypocotyl elongation, MeIAA was much more potent than IAA in a hypocotyl elongation assay, indicating that IAA activities could be effectively regulated by methylation. IAMT1 was spatially and temporally regulated during the development of both rosette and cauline leaves. Changing expression patterns and/or levels of IAMT1 often led to dramatic leaf curvature phenotypes. In iamt1-D, the decreased expression levels of TCP genes, which are known to regulate leaf curvature, may partially account for the curly leaf phenotype. The identification of IAMT1 and the elucidation of its role in Arabidopsis leaf development have broad implications for auxin-regulated developmental process.

Disruption of phytoene desaturase gene results in albino and dwarf phenotypes in Arabidopsis by impairing chlorophyll, carotenoid, and gibberellin biosynthesis.

Carotenoids play an important role in many physiological processes in plants and the phytoene desaturase gene (PDS3) encodes one of the important enzymes in the carotenoid biosynthesis pathway. Here we report the identification and analysis of a T-DNA insertion mutant of PDS3 gene. Functional complementation confirmed that both the albino and dwarf phenotypes of the pds3 mutant resulted from functional disruption of the PDS3 gene. Chloroplast development was arrested at the proplastid stage in the pds3 mutant. Further analysis showed that high level of phytoene was accumulated in the pds3 mutant. Addition of exogenous GA3 could partially rescue the dwarf phenotype, suggesting that the dwarf phenotype of the pds3 mutant might be due to GA deficiency. Microarray and RT-PCR analysis showed that disrupting PDS3 gene resulted in gene expression changes involved in at least 20 metabolic pathways, including the inhibition of many genes in carotenoid, chlorophyll, and GA biosynthesis pathways. Our data suggest that the accumulated phytoene in the pds3 mutant might play an important role in certain negative feedbacks to affect gene expression of diverse cellular pathways.

Targeted Degradation of the Cyclin-Dependent Kinase Inhibitor ICK4/KRP6 by RING-Type E3 Ligases Is Essential for Mitotic Cell Cycle Progression during Arabidopsis Gametogenesis

Following meiosis, plant gametophytes develop through two or three rounds of mitosis. Although the ontogeny of gametophyte development has been defined in Arabidopsis thaliana, the molecular mechanisms regulating mitotic cell cycle progression are not well understood. Here, we report that RING-H2 group F 1a (RHF1a) and RHF2a, two RING-finger E3 ligases, play an important role in Arabidopsis gametogenesis. The rhf1a rhf2a double mutants are defective in the formation of male and female gametophytes due to interphase arrest of the mitotic cell cycle at the microspore stage of pollen development and at female gametophyte stage 1 of embryo sac development. We demonstrate that RHF1a directly interacts with and targets a cyclin-dependent kinase inhibitor ICK4/KRP6 (for Interactors of Cdc2 Kinase 4/Kip-related protein 6) for proteasome-mediated degradation. Inactivation of the two redundant RHF genes leads to the accumulation of ICK4/KRP6, and reduction of ICK4/KRP6 expression largely rescues the gametophytic defects in rhf1a rhf2a double mutants, indicating that ICK4/KRP6 is a substrate of the RHF E3 ligases. Interestingly, in situ hybridization showed that ICK4/KRP6 was predominantly expressed in sporophytes during meiosis. Our findings indicate that RHF1a/2a-mediated degradation of the meiosis-accumulated ICK4/KRP6 is essential to ensure the progression of subsequent mitoses to form gametophytes in Arabidopsis.

NPY1, a BTB-NPH3-like protein, plays a critical role in auxin-regulated organogenesis in Arabidopsis.

Auxin is an essential regulator for plant development. To elucidate the mechanisms by which auxin regulates plant development, we isolated an Arabidopsis mutant naked pins in yuc mutants 1 (npy1) that develops pin-like inflorescences and fails to initiate any flowers in yuc1 yuc4, a background that is defective in auxin biosynthesis. The phenotypes of npy1 yuc1 yuc4 triple mutants closely resemble those of Arabidopsis mutants pin-formed1 (pin1), pinoid (pid), and monopteros (mp), which are defective in either auxin transport or auxin signaling. NPY1 belongs to a large family of proteins and is homologous to NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3), a BTB/POZ protein that regulates phototropic responses along with the protein kinase PHOT1 (Phototropin 1). We demonstrate that NPY1 works with the protein kinase PID, which is homologous to PHOT1, to regulate auxin-mediated plant development. The npy1 pid double mutants fail to form any cotyledons, a phenotype that is also observed in yuc1 yuc4 pid triple mutants. Interestingly, both auxin-regulated organogenesis and phototropic responses require an auxin response factor (ARF). Disruption of ARF7/NPH4 leads to nonphototropic hypocotyls and arf5/mp forms pin-like inflorescences. Whereas the PHOT1/NPH3 pathway is regulated by light, our data suggest that the PID/NPY1 pathway may be regulated by auxin synthesized by the YUC flavin monooxygenases. Our findings put YUCs, PID, and NPY1 into a genetic framework for further dissecting the mechanisms of auxin action in plant development.

Dof5.6/HCA2 , a Dof Transcription Factor Gene, Regulates Interfascicular Cambium Formation and Vascular Tissue Development in Arabidopsis

Vascular cambium, a type of lateral meristem, is the source of secondary xylem and secondary phloem, but little is known about the molecular mechanisms of its formation and development. Here, we report the characterization of an Arabidopsis thaliana gain-of-function mutant with dramatically increased cambial activity, designated high cambial activity2 (hca2). The hca2 mutant has no alternative organization of the vascular bundles/fibers in inflorescence stems, due to precocious formation of interfascicular cambium and its subsequent cell division. The phenotype results from elevated expression of HCA2, which encodes a nuclear-localized DNA binding with one finger (Dof) transcription factor Dof5.6. Dof5.6/HCA2 is preferentially expressed in the vasculature of all the organs, particularly in the cambium, phloem, and interfascicular parenchyma cells of inflorescence stems. Dominant-negative analysis further demonstrated that both ubiquitous and in situ repression of HCA2 activity led to disruption of interfascicular cambium formation and development in inflorescence stems. In-depth anatomical analysis showed that HCA2 promotes interfascicular cambium formation at a very early stage of inflorescence stem development. This report demonstrates that a transcription factor gene, HCA2, is involved in regulation of interfascicular cambium formation and vascular tissue development in Arabidopsis.

CFL1, a WW Domain Protein, Regulates Cuticle Development by Modulating the Function of HDG1, a Class IV Homeodomain Transcription Factor, in Rice and Arabidopsis

This study identifies CFL1 as a negative regulator of cuticle development in rice and Arabidopsis. CFL1 interacts with a class IV homeodomain-leucine zipper transcription factor to modulate the expression of two genes known to be involved in cuticle development. Plants have a chemically heterogeneous lipophilic layer, the cuticle, which protects them from biotic and abiotic stresses. The mechanisms that regulate cuticle development are poorly understood. We identified a rice (Oryza sativa) dominant curly leaf mutant, curly flag leaf1 (cfl1), and cloned CFL1, which encodes a WW domain protein. We overexpressed both rice and Arabidopsis CFL1 in Arabidopsis thaliana; these transgenic plants showed severely impaired cuticle development, similar to that in cfl1 rice. Reduced expression of At CFL1 resulted in reinforcement of cuticle structure. At CFL1 was predominantly expressed in specialized epidermal cells and in regions where dehiscence and abscission occur. Biochemical evidence showed that At CFL1 interacts with HDG1, a class IV homeodomain-leucine zipper transcription factor. Suppression of HDG1 function resulted in similar defective cuticle phenotypes in wild-type Arabidopsis but much alleviated phenotypes in At cfl1-1 mutants. The expression of two cuticle development-associated genes, BDG and FDH, was downregulated in At CFL1 overexpressor and HDG1 suppression plants. HDG1 binds to the cis-element L1 box, which exists in the regulatory regions of BDG and FDH. Our results suggest that rice and Arabidopsis CFL1 negatively regulate cuticle development by affecting the function of HDG1, which regulates the downstream genes BDG and FDH.

Arabidopsis AtBECLIN 1/AtAtg6/AtVps30 is essential for pollen germination and plant development.

Pollen germination on the surface of compatible stigmatic tissues is an essential step for plant fertilization. Here we report that the Arabidopsis mutant bcl1 is male sterile as a result of the failure of pollen germination. We show that the bcl1 mutant allele cannot be transmitted by male gametophytes and no homozygous bcl1 mutants were obtained. Analysis of pollen developmental stages indicates that the bcl1 mutation affects pollen germination but not pollen maturation. Molecular analysis demonstrates that the failure of pollen germination was caused by the disruption of AtBECLIN 1. AtBECLIN 1 is expressed predominantly in mature pollen and encodes a protein with significant homology to Beclin1/Atg6/Vps30 required for the processes of autophagy and vacuolar protein sorting (VPS) in yeast. We also show that AtBECLIN 1 is required for normal plant development, and that genes related to autophagy, VPS and the glycosylphosphatidylinositol anchor system, were affected by the deficiency of AtBECLIN 1.